Nanofraction immune modulators, preparations and compositions including the same, and associated methods

ABSTRACT

Compositions that include extracts from sources of immune modulators that include nanofraction immune modulator molecules (i.e., molecules having molecular weights of about 3,000 Da and less) are disclosed. These compositions may also include other immune modulators, such as transfer factor. Administration of compositions with extracts that include nanofraction immune modulator molecules modulates the cell-mediated immunity (e.g., down-regulates undesired T cell activity) of a subject to which such compositions are administered. When administered with transfer factor, the combination of nanofraction immune modulator molecules and transfer factor down-regulates undesired T cell activity while increasing, or up-regulating, T cell activity against pathogens and other undesirable entities, such as cancer cells and other aberrant or mutated cells. Assays and assay techniques for evaluating the immune modulation capabilities of various substances are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/855,944, filed Sep. 14, 2007, titled NANOFRACTION IMMUNE MODULATORS,PREPARATIONS AND COMPOSITIONS INCLUDING THE SAME, AND ASSOCIATED METHODS(“the '944 Application”), now U.S. Pat. No. 10,471,100, issued Nov. 12,2019. The '944 Application includes a claim for priority to the Sep. 29,2006 filing date of U.S. Provisional Patent Application No. 60/848,348(“the '348 Provisional Application”) under 35 U.S.C. § 119(e). Theentire disclosures of the '348 Provisional Application and the '944Application are hereby incorporated herein.

TECHNICAL FIELD

The present invention relates to molecules that modulate (e.g., elicit,enhance, suppress undesirable activity, etc.) cell-mediated immunity ina subject, including methods for generating and obtaining suchmolecules, preparations and compositions that include such molecules,methods for evaluating the effectiveness of such molecules, and methodsof use. More specifically, the present invention relates to smallmolecules, which are referred to herein as “nanofraction” molecules thatmodulate cell-mediated immunity.

RELATED ART

The ability of antibodies to provide and transfer immunity is well knownand widely researched, as are the characteristics of antibodies and themechanisms by which antibodies are produced.

Not so well known or so widely researched are the roles transferfactors, which includes a family of molecules having molecular weightsof between 3,500 Da and 7,500 Da, play in modulating cellular, orT-cell-mediated, immunity. Over time, the understanding that those ofskill in the pertinent art have about the characteristics of transferfactors and their roles in an organism's immune system has improved andcontinues to improve.

While further research continues to shed light on the characteristicsand functions of a wide variety of immune system components, there maybe a large number of poorly understood, or even overlooked moleculesthat may have an impact on the manner in which immunity is developed,maintained, conveyed, and transferred, as well as on the effects ofimmunity on longevity.

SUMMARY

The effectiveness of various molecules in modulating cell-mediatedimmunity has recently been characterized in a quantifiable manner.Molecules that may directly or indirectly modulate cell-mediatedimmunity are known in the art as “immune modulators.” One class ofimmune modulators includes small, or low molecular weight, nanofraction(e.g., up to 3,000 Da, up to 3,500 Da, 250 Da to 2,000 Da, 2,000 Da to4,000 Da) molecules that elicit, enhance, suppress, or otherwisemodulate a cell-mediated immune response. Due to the relatively smallsizes, or molecular weights, of such immune modulators, they arereferred to herein as “nanofraction” immune modulators and as“nanofraction” molecules.

Nanofraction immune modulators may be obtained from a variety ofdifferent types of source animals. Examples of source animals include,but are not limited to, mammals (e.g., cows) and birds (e.g., chickens).Without limiting the scope of the present invention, nanofraction immunemodulators may be obtained from colostrum, or even milk, produced by amammal. As another non-limiting example, nanofraction immune modulatorsmay be acquired from eggs produced by birds or any other type of animal.Colostrum, eggs, and other sources of nanofraction molecules arecollectively referred to herein as “nanofraction sources.”

The natural production of nanofraction immune modulators by a sourceanimal may be enhanced by exposing the source animal to a greateramount, or concentration, of one or more antigens than the amount(s) ofsuch antigen(s) to which the source animal would normally be exposed.For example, if a particular type of source animal, or even a specificsource animal, would, in its typical environment, normally be exposed toa certain amount or concentration of a given antigen, the sourceanimal's production of immune modulators, including nanofractionmolecules, may be enhanced by exposing the source animal to an evengreater amount (e.g., concentration) of that antigen (e.g., byvaccinating the source animal, by placing the source animal into anenvironment where a greater amount or concentration of that antigen ispresent, etc.). As another example, if a particular type of sourceanimal, or even a specific source animal, were typically vaccinated witha given antigen, the source animal's production of one or morenanofraction immune modulators could be enhanced by increasing theexposure of the source animal to an antigen (e.g., by exposing thesource animal to an increased concentration of the antigen, a moreeffective or more virulent form of the antigen, etc.), although thenanofraction molecules are not themselves believed to be antigenspecific.

Known processes may be used to partially, substantially, or completelypurify nanofraction immune modulators from other molecules present inthe nanofraction source animal from which they are obtained and,optionally, to concentrate the nanofraction immune modulators. Suchprocesses include, without limitation, mechanical separation, phaseseparation (e.g., separation of aqueous and non-aqueous components fromone another), precipitation, centrifugation, filtration (includingmicrofiltration, with a molecule weight cutoff (MWCO) in the range ofabout 12,000 Da down to about 4,000 Da, and nanofiltration, with an MWCOof less than about 4,000 Da), dialysis, chromatographic, andelectrophoretic purification processes. Such processes may be effectedindividually or in any combination to produce a preparation in which oneor more types of immune modulators are present.

In one aspect, the present invention includes preparations of at leastpartially purified, substantially purified (e.g., to a degree acceptedby those in the pertinent art), and completely purified immunemodulators. Additionally, the present invention includes compositionsthat include nanofraction molecules. In addition to nanofractionmolecules, such compositions may include other components that areuseful in supporting or modulating the immune system of a subject (e.g.,transfer factor, antibodies, etc.), as well as components that maybenefit the subject in other ways.

Methods that include use or administration of nanofraction molecules orcompositions including the same, alone or with other immune modulators,are also within the scope of the present invention. Methods of useinclude the administration of one or more types of immune modulators(e.g., in raw, partially purified, substantially purified, or completelypurified form, in a preparation, in a composition, etc.) to a subject(e.g., a human or any type of animal that is believed to benefit fromthe immune modulation provided by nanofraction molecules). The immunemodulators are administered to a subject in an amount that increases thelevel (e.g., concentration) of a particular, administered type of immunemodulator in the body of the subject to an above-normal amount for thesubject. Without limiting the scope of this aspect of the presentinvention, a subject may receive an amount of one or more immunemodulators that is clinically effective for causing the immune system ofthe subject to elicit a cell-mediated immune response or an amount thateffectively enhances a cell-mediated immune response by the subject.

In addition, tests and testing methods that evaluate the effectivenessof immune modulators are within the scope of the present invention. Asan example, a T-cell immune function assay may be used to evaluate theability of a potential immune modulator to modulate the activity of(e.g., production of adenosine tri-phosphate (ATP) by) one or more typesof cells that participate in cell-mediated immunity, either alone or inconjunction with other molecules (e.g., antigens, mitogens (which inducemitosis, or cell replication, etc.).

Other features and advantages will become apparent to those of skill inthe art through consideration of the ensuing description and theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, FIGS. 1 through 4 are graphic illustrations of theresults of various tests performed on compositions that incorporateteachings of the present invention.

DETAILED DESCRIPTION

It has recently been discovered that small molecules in a variety ofmolecular weight ranges are useful in modulating the activity of immunecells. The following EXAMPLES set forth the acts that were performed toreach these conclusions.

Example 1

Using known processes, including phase separation, precipitation,filtration, microfiltration or nanofiltration, and dialysis, a varietyof molecular weight fractions were prepared from both bovine colostrumand chicken eggs. The molecular weight fractions that were obtained frombovine colostrum were: 250 Da to 2,000 Da, 2,000 Da to 4,000 Da, 4,000Da to 8,000 Da (which includes transfer factor, and was included for thesake of comparison), and 8,000 Da to 12,000 Da. Similarly, 2,000 Da to4,000 Da, 4,000 Da to 8,000 Da (which includes transfer factor, and wasincluded for the sake of comparison), and 8,000 Da to 12,000 Damolecular weight fractions were prepared from chicken egg yolks. Themolecular weight fractions were then dried to powder form (e.g., byspray drying, freeze drying, etc.).

Various assays were then conducted using these preparations to evaluatethe effects of molecules in each fraction to modulate the activity ofcells that convey cellular immunity (e.g., CD4+ T helper cells).Specifically, assays of the type disclosed in U.S. Pat. Nos. 5,773,232and 6,630,316 and in U.S. Patent Application Publication 2005/0260563,the entire disclosure of each of which is hereby incorporated herein, inits entirety, by this reference, were modified and used to evaluate theactivities of different molecular weight fractions from EXAMPLE 1 in avariety of conditions. The aforementioned assays are used to evaluatethe production of adenosine tri-phosphate (ATP) by immune cells (e.g.,CD4+ T helper cells, CD3+ cells (which includes all T-cells), etc.). Theamount of ATP produced by the cells may be measured in a manner known inthe art (e.g., by use of the so-called “Luciferin reaction” with alumenometer).

Example 2

A first series of assays was conducted using the white blood cells ofhealthy individuals that include, or express, so-called “CD4”glycoproteins on their surfaces using the ImmuKnow™ assay produced byCylex Incorporated of Columbia, Md. These white blood cells are alsoreferred to as “CD4+” cells due to their expression of the CD4glycoproteins. Expression of the CD4 glycoprotein distinguishesso-called “T helper” cells from other types of white blood cells,including other T-cells.

The components of the ImmuKnow™ assay test kit were: a standard 96-well“Assay Plate,” including removable eight well strips; a “SampleDiluent,” which includes growth medium and a preservative; a“Stimulant,” which includes phytohemagglutinin-L (PHA-L) (a substancefrom beans (e.g., red kidney beans) that is known to nonspecificallystimulate mitosis (a process in which a cell grows and splits into twonew cells) and, thus, the preparatory production of adenosinetri-phosphate (ATP) in white blood cells (i.e., a “mitogen”)),introduced into the remaining four “stimulated” wells of the eight wellstrip, diluted in growth medium and a preservative; “Dynabeads®* CD4,”which are magnetic sample purification beads coated with mousemonoclonal anti-human CD4 antibodies and are carried by a bufferedsaline solution with bovine serum albumin (BSA) and preservative; a“Wash Buffer,” which includes a buffered saline solution with BSA; a“Lysis Reagent,” which includes a hypotonic basic solution withdetergent, a “Calibrator Panel” with ATP concentrations of 0, 1, 10,100, and 1,000 ng/ml; a “Luminescence Reagent” including luciferin andluciferase in a buffered solution, which reacts with ATP to create lightin an amount indicative of the amount of ATP to which the LuminescenceReagent has been exposed; and a “Measurement Plate” with 96 wells thathave opaque boundaries (i.e., walls and bases).

One eight well strip of the 96 well Assay Plate, which may be referredto as a “control strip,” was used to provide controls, including four“nonstimulated” (NS) control wells and four “stimulated” control wells.

Another eight well strip of a 96 well Assay Plate, which may be referredto as a “test strip,” was used for each sample to be tested. Four of thewells of each strip were designated as “nonstimulated” wells, while theother four wells of each strip were “stimulated” wells. Fiftymicroliters (50 μl) of Sample Diluent was introduced into each of thefour “nonstimulated” wells of the control strip, while 25 μl of SampleDiluent was introduced into each of the “nonstimulated” wells of eachtest strip. Twenty-five microliters (25 μl) of Stimulant was introducedinto each of the four “stimulated” wells of the control strip and intoeach of the four “stimulated” wells of each test strip.

In addition to the Sample Diluent or Stimulant, a 25 μl sample of one ofthe molecular weight fractions identified in EXAMPLE 1 was introducedinto each of the eight wells of each test strip. More specifically, eachof the molecular weight fractions of EXAMPLE 1 was reconstituted inSample Diluent and diluted with a volume of Sample Diluent to providethree different concentrations that would ultimately, upon addition of a25 μl sample to a well, respectively amount to the addition of 10 μg,100 μg, and 1,000 μg of the dried powder to the well.

A 1:3 (blood: “Sample Diluent”) dilution of a blood sample, which hadbeen gently agitated to uniformly distribute its constituents, includingwhite blood cells, was prepared. Seventy-five microliters (75 μl) ofdiluted blood was added to each well of each strip. The contents of thewells were then mixed (e.g., by placing the plate on a shaker plate forabout 30 seconds), then incubated at a temperature of 37° C. in a 5% CO₂environment for about 18 hours.

Once incubation was complete, the contents of the wells were again mixed(e.g., by placing the plate on a shaker plate for about three minutes).Thereafter, the solution including the Dynabeads® sample purificationbeads was mixed to homogenously suspend the Dynabeads® samplepurification beads within the liquid by which they were carried (e.g.,with a vortex). As noted above, the Dynabeads® sample purification beadsin this example include magnetic beads coated with mouse monoclonalanti-human CD4 antibodies. Fifty microliters (50 μl) of the Dynabead®sample purification bead-carrying solution was added to each well ofeach strip.

The contents of the wells of each strip were again mixed (e.g., on ashaker plate for about 15 seconds), then allowed to set, or incubate, atroom temperature for a duration of about 15 minutes. The process ofmixing and incubation was then repeated. The mouse anti-human CD4antibodies on the Dynabeads® sample purification beads bind only towhite blood cells exhibiting the CD4 glycoprotein. During thisincubation, CD4+ white blood cells, which include T helper cells, wereimmobilized by, or bound to, the mouse monoclonal anti-human CD4antibodies on the Dynabeads® sample purification beads.

Following incubation, the contents of each well were mixed again (e.g.,for about 15 seconds to about 30 seconds on a shaker plate) to resuspendthe Dynabeads® sample purification beads. The contents of each well werethen introduced into a magnetic field (e.g., by placing each eight wellstrip in a magnet tray available from Cylex), in accordance with theprotocol set forth in the instructions that accompany the ImmuKnow™assay. When subjected to the magnetic field, the Dynabeads® samplepurification beads are pulled to one side of each well in which they arepresent. The remaining contents of the well may then be removed (e.g.,aspirated with a pipette, etc.) and the beads and T helper cells washedone or more times (e.g., three times, each with 200 μl Wash Buffer) tosubstantially purify the same.

Two hundred microliters (200 μl) of Lysis Reagent was then added to eachwell. Following removal of the contents of each well from the magneticfield, the contents of each well (i.e., the Dynabeads® samplepurification beads, cells attached thereto, and the Lysis Reagent) weremixed (e.g., for about five minutes on a plate shaker). The LysisReagent disrupted the membranes of the CD4+ cells that were immobilizedby antibody on the Dynabeads® sample purification beads. Among otherthings, ATP was released from the lysed cells.

Once cell lysis was complete, the contents of each well were againsubjected to a magnetic field, pulling the Dynabeads® samplepurification beads within each well to one side of the well. A 50 μlsample was then transferred from each well to a corresponding well ofthe Measurement Plate. In addition to transferring samples, severalwells of the 96 well Measurement Plate were reserved for 50 μl samplesof the various ATP concentrations of the Calibrator Panel solutions.

One hundred fifty microliters (150 μl) of the Luminescence Reagent wasthen added to each well of the Measurement Plate that included either atest sample or a sample of Calibrator Panel solution. The luminescencefrom each well was then measured. The measured luminescence from eachwell provides an indication of the amount of ATP present in that well.The amount of ATP present in each well is, in turn, indicative of anamount of metabolic activity within the cells (i.e., CD4+ cells) fromwhich the contents of each well of the Measurement Plate. In the samplesthat were derived from cells that were nonspecifically stimulated withPHA, a relatively high level of ATP is expected to be present. Theaddition of an immune modulator (e.g., from one of the fractionsidentified in EXAMPLE 1) would increase or decrease, or modulate, themetabolic activity in CD4+ cells that was nonspecifically stimulated byPHA.

The results of such testing are set forth in the following table, withthe illustrated numbers, representing the mean (average) amount of ATPproduced by the white blood cells of each subset:

TABLE 1 10 μg 100 μg 1000 μg Sample Control per well per well per well250 Da to Non-Stimulated 14 52 50 37 2,000 Da (NS) Colostrum Stimulatedwith 388 336 253 127 fraction PHA % reduction in 13.4 34.8 67.3 PHAstimulation 2,000 Da to NS 14 52 62 42 4,000 Da Stimulated with 388 388377 339 Colostrum PHA fraction % reduction in 0 2.8 12.6 PHA stimulation4,000 Da to NS 14 69 50 46 8,000 Da Stimulated with 388 378 250 207(includes TF) PHA Colostrum % reduction in 2.6 35.6 46.6 fraction PHAstimulation 8,000 Da to NS 14 49 45 39 12,000 Da Stimulated with 388 337237 181 Colostrum PHA fraction % reduction in 13.1 38.9 53.4 PHAstimulation 2,000 Da to NS 14 49 33 44 4,000 Da Stimulated with 388 228161 148 Egg fraction PHA % reduction in 41.2 58.5 61.9 PHA stimulation4,000 Da to NS 14 54 47 44 8,000 Da Stimulated with 388 354 230 158(includes TF) PHA Egg fraction % reduction in 8.8 40.7 59.3 PHAstimulation

These data show that in the non-stimulated tests, where cells were notexposed to PHA, each of the 4,000 Da to 8,000 Da molecular weightfractions (from colostrum and egg), both of which are known to containtransfer factor, stimulated additional metabolic activity in the CD4+white blood cells. These data confirm the ability of transfer factor toup-regulate cell-mediated immunity.

Conversely, the 4,000 Da to 8,000 Da colostrum and egg fractionsdown-regulated the nonspecific ability of PHA to stimulate metabolicactivity in CD4+ cells. Since PHA is an artificial, nonspecificstimulant, the down-regulation of its activity by transfer factor, whichparticipates in cell-mediated immunity, is not surprising. It isbelieved, and previous research has shown, that transfer factor helpsbalance, and even focus, immune activity by T-cells (e.g., by helpingthe cells “remember” their primary purpose, by reducing autoimmunity andassociated disorders, while improving activity against undesirableentities, such as infection of a subject's body by microorganisms(bacteria, viruses, etc.), etc.). The down-regulation of PHA-stimulatedactivity by T-cells appears to confirm this role of transfer factor incell-mediated immunity.

Similar results were seen in a number of other molecular weightfractions that do not include transfer factor, including the 250 Da to2,000 Da colostrum fraction (both up-regulation and down regulation),the 2,000 Da to 4,000 Da colostrum fraction (up-regulation), and the2,000 Da to 4,000 Da egg fraction (up-regulation and down-regulation).The 8,000 Da to 10,000 Da colostrum fraction also caused up-regulationof activity in CD4+ white blood cells that were not stimulated with PHAand down-regulation of PHA stimulation of metabolic activity in CD4+white blood cells.

These data establish that immune modulators other than transfer factorare present in at least the 250 Da to 2,000 Da colostrum fraction, the2,000 Da to 4,000 Da colostrum fraction, and the 2,000 Da to 4,000 Daegg fraction. The immune modulation capabilities of the “nanofractionmolecules” present in each of these fractions has been at leastpartially confirmed by the experiment that is set forth in EXAMPLE 3.

Example 3

A second series of assays of activity induced in a healthy individual'swhite blood cells exhibiting the CD3 glycoprotein (i.e., CD3+ cells),which are known to include all T-cells, including so-called “T memory”cells. Specifically, Cylex's T-Cell Memory™ assay was used. The protocolof Cylex's T-Cell Memory™ assay is very similar to that set forth inEXAMPLE 2, with the following exceptions: 25 μl of the Stimulant, whichincluded Concanavalin A (ConA) instead of PHA, was only introduced intothe “stimulated” wells of the control strip, while 25 μl of a 1:10dilution of cytomegalovirus (CMV) vaccine was added to the “stimulated”wells of each test strip (for a final, per well dilution of 1:50); mouseanti-human CD3 antibodies were immobilized to the surfaces of magneticbeads (per instructions accompanying the T-Cell Memory™ test kit) toprepare the Dynabeads® sample purification beads; and the Dynabeads®sample purification beads were added to the blood samples, SampleDiluent, Stimulant (if any), and sample fraction (if any) before theinitial incubation.

In the T-Cell Memory™ assay, antigen is used in place of a mitogen(e.g., PHA) so that the ability of the T memory cells to recognize aparticular antigen may be evaluated. Notably, the intensity of the lightemitted from each well of the Measurement Plate is less, as T memorycells make up only a portion of the cells that have been bound toantibody molecules of the Dynabeads®.

The results of these assays are set forth in the following table, withthe illustrated numbers representing the mean (average) amount of ATPproduced by the white blood cells for each sample (and amount) tested:

TABLE 2 Sample Control 10 μg 100 μg 1000 μg 250 Da to Non-Stimulated 1214 12 17 2,000 Da (NS) Colostrum Stimulated with 316 468 338 345fraction CMV % increase in 48.1 7.0 9.2 CMV stimulation 2,000 Da to NS12 15 12 15 4,000 Da Stimulated with 316 501 503 440 Colostrum CMVfraction % increase in 58.5 59.1 39.2 CMV stimulation 4,000 Da to NS 1222 16 19 8,000 Da Stimulated with 316 473 476 475 (includes TF) CMVColostrum % increase in 49.7 50.6 50.3 fraction CMV stimulation 8,000 Dato NS 12 14 18 26 12,000 Da Stimulated with 316 453 404 370 ColostrumCMV fraction % increase in 43.4 27.8 17.1 CMV stimulation 2,000 Da to NS12 26 61 108 4,000 Da Stimulated with 316 305 349 350 Egg fraction CMV %increase in −3.5 10.4 10.8 CMV stimulation 4,000 Da to NS 12 34 39 1088,000 Da Stimulated with 316 310 280 381 (includes TF) CMV Egg fraction% increase in −1.9 −11.4 20.6 CMV stimulation

The data obtained from the testing conducted in this EXAMPLE 3demonstrate that, in the presence of an antigen (i.e., a specificstimulant, as opposed to the nonspecificity of a mitogen, such as ConAor PHA), the three non-transfer factor-containing colostral fractionsenhance the activity of the tested T memory cells to CMV to a degreethat is comparable to (the 10 μg samples of the 250 Da to 2,000 Da and8,000 Da to 12,000 Da colostrum fractions) or exceeds (the 10 μg and 100μg samples of the 2,000 Da to 4,000 Da colostrum fraction) the abilityof the comparably sized samples of the 4,000 Da to 8,000 Da, transferfactor-containing colostrum fraction to enhance the activity of thetested cells when exposed to CMV.

Example 4

Another set of assays was conducted to determine whether or not eitherthe nanofraction immune modulator molecules (i.e., the immune modulatorsof the 2,000 Da to 4,000 Da colostrum fraction) or the transfer-factorcontaining fraction (i.e., the 4,000 Da to 8,000 Da colostrum fraction)could modulate (e.g., enhance) the immune memory of a subject who hadrecently been exposed to a high dose of a particular antigen.Specifically, a blood sample was obtained from an individual who hadbeen exposed to an influenza virus, which causes a systemic infection,and suffered from influenza symptoms for four weeks.

The assay was conducted in the manner described in EXAMPLE 3, using theCylex T-Cell Memory™ assay in accordance with the instructions providedwith that assay and set forth in EXAMPLE 3, except an influenza antigen,in the form of 1:25 dilution of the influenza vaccine manufactured byAventis Pasteur of Paris, France, for the 2006-2007 flu season (for afinal, per well dilution of 1:125), was used in place of the CMV vaccineof EXAMPLE 3.

The results from that assay are set forth in the following table:

TABLE 3 Sample Control 10 μg 100 μg 1000 μg 2,000 Da to NS 4 10 3 44,000 Da Stimulated with 827 1003 906 936 Colostrum influenza antigenfraction ConA 694 % increase in 21.3 9.6 13.2 influenza antigenstimulation 4,000 Da to NS 4 36 24 11 8,000 Da Stimulated with 827 989997 830 (includes TF) influenza antigen Colostrum ConA 694 fraction %increase in 19.6 20.6 0.4 influenza antigen stimulation

The results shown in TABLE 3 (which are also shown graphically inFIG. 1) indicate that, when T memory cells of a subject who has recentlybeen exposed to a particular antigen are exposed to that antigen,particularly in the presence of nanofraction molecules or transferfactor, activity of the CD3+ memory T-cells increases significantly. Infact, relatively small amounts of nanofraction molecules and of transferfactor caused an increase of about 20% in T memory cell activity. Infact, it appears that the immune modulators of the 2,000 Da to 4,000 Dafraction are about as effective as the transfer factor and any othermolecules present in the 4,000 Da to 8,000 Da fraction in modulating theactivity of the tested cells.

The results from EXAMPLES 1-4 indicate that immune modulators havingmolecular weights in the 250 Da to 2,000 Da, 2,000 Da to 4,000 Da, and8,000 Da to 12,000 Da ranges are effective in modulating immune activityof various types of T-cells. Thus, by administering such immunemodulators or preparations or compositions including the immunemodulators to a subject, the subject's cell-mediated immunity may bemodulated.

Based on these results, a process was developed for producing variousdietary supplements (e.g., from (bovine) colostrum, (chicken) egg, etc.)that include molecules of a predetermined MWCO. For example, and not byway of limitation, a liquid preparation of a source of nanofractionimmune modulators, from which at least macroscopic particles (e.g.,colostrum/milk solids, egg shells and membranes, etc.) (e.g., by phaseseparation, filtration processes, etc.) have been removed may be forcedthrough a filter with pores that are sized to provide the predeterminedupper MWCO. As a nonlimiting example, a filter that provides a molecularweight cutoff of about 3,000 Da may be used. Alternatively, dialysisprocesses, which include use of dialysis membranes having pores thatprovide the desired MWCO, may be used. The use of such processesprovides a “nanofraction” from which larger molecules, includingtransfer factor, antibodies, and a variety of other molecules havingmolecular weights of greater than about 3,000 Da, are excluded (e.g.,colostrum, chicken and various powdered compositions were produced. Thefiltrate (i.e., the portion of the liquid that has passed through thefilter) may then be further processed by known techniques (e.g., freezedrying, spray drying, evaporation to form a more concentrated liquid,incorporation into a gel, etc.). The resulting “nanofraction product”may then be used alone or incorporated into other compositions.

It is believed that by including nanofraction molecules, even in verysmall amounts, in preparations that also include transfer factor (andwhich may also include baseline levels (i.e., those levels alreadypresent in the source (e.g., colostrum, egg, etc.) from which transferfactor is obtained), the resulting compositions will down-regulateundesired activity by T-cells (e.g., autoimmunity and associateddisorders, etc.), while improving, or up-regulating, desired T-cellactivity. The nanofraction-and-transfer factor compositions that are setforth in TABLES 4 and 5 were developed.

TABLE 4 COMPOSITION A Relative Amount Ingredient (by weight) BovineColostrum fraction, upper MWCO 10,000 Da 68% (spray dried) BovineColostrum fraction, upper MWCO 3,000 Da  2% (nanofraction) (spray dried)Chicken Egg Yolk (spray dried) 30%

The composition of TABLE 4 may also be referred to as an “immunemodulating component.” Such an “immune modulating component” may consistessentially of a combination of sources of immune modulators (includingsources of nanofraction immune modulators) or extracts of immunemodulator sources, such as those listed in TABLE 4, or it may includeother ingredients.

Likewise, a composition that incorporates teachings of the presentinvention may consist essentially of an “immune modulating component,”such as that disclosed in TABLE 4, or it may include other ingredients,as set forth in TABLE 5.

TABLE 5 COMPOSITION B Amount (per serving, serving size = one Ingredientcapsule) Composition A 150 mg Zinc (as monomethionine)  5 mg Cordyvant ™Proprietary Polysaccharide Complex 440 mg IP-6 (Inositol hexaphosphate)Soya bean Extract (phytosterols) Cordyceps sinensis (7% cordycepticacids) Beta-Glucan (from baker's yeast) (Saacharomyces cerevisiae)Beta-Glucan (from Oat) (Avena sativa) Agaricus blazeii Extract Mannans(from Aloe vera) (leaf) Olive Leaf Extract (Olea europaea) MaitakeMushroom (Grifola frondosa) (whole plant) Shiitake Mushroom (Lentinusedodes) (whole plant) (5:1 extract)

A composition according to the present invention may be embodied as aliquid (e.g., into the RioVida® drink available from 4Life Research, LC,of Sandy, Utah), a powder (which may include additional ingredients toprovide a desirable flavor, dissolution properties, and the like), atablet (which additionally includes other ingredients, such as binders(e.g., starch) and the like, a gel (in which gelatin or otheringredients may be added), or in any other suitable form. It should beunderstood that, for purposes of this disclosure, the additionalingredients that are used to manufacture such embodiments of acomposition of the present invention may, for purposes of thisdisclosure, merely be considered to be optional and nonessential to thecomposition, unless otherwise required by an appended claim.

Example 5

Blood was collected from an individual who had been suffering fromshingles (varicella zoster virus (VZV) infection) symptoms for aboutfour weeks. The blood was then assayed using the ImmuKnow™ assay in themanner described in EXAMPLE 2, with the sample fractions of EXAMPLE 2having been replaced with the following: (a) a control that included noimmune modulators; (b) a colostrum fraction having a MWCO of about 3,000Da that had been spray dried; (c) Transfer Factor XF®, which iscurrently available from 4Life Research and includes a bovine colostrumextract with an upper MWCO of about 10,000 Da, was added; (d) thecomposition in TABLE 4, which is labeled as “Composition A”; and (e) thecomposition of TABLE 5, which is labeled as “Composition B.” Each of (b)through (e) was reconstituted in the Sample Diluent that accompanied theImmuKnow™ assay, and diluted to a concentration that resulted in afinal, per-well concentration of 1 mg/ml once blood samples and allother liquids had been added to each well.

The results from those assays are set forth in the following table:

TABLE 6 Nano- TF Composi- Composi- Control fraction XF tion A tion BNon-Stimulated (NS) 26 35 77 47 19 Stimulated with PHA 220 234 150 14027

These results also appear in the graph of FIG. 2.

It is reiterated that these results were obtained at a point in time(about four weeks following initial infection; i.e., duringconvalescence) where, in the absence of stimulation, T helper (CD4+)cell activity is expected to decrease, although a large number of Thelper cells remain in the subject's blood. T helper cell activity wasstimulated only slightly, in the absence of the nonspecific stimulantPHA, by the nanofraction, TF XF, and Composition A, and does not appearto have been stimulated by Composition B. The nonspecific stimulation ofT helper cells by PHA was, however, reduced significantly by the TF XFand Composition A, and to an even larger extent by the Composition B, asmight be expected from the results of EXAMPLE 2, as set forth in TABLE1.

Example 6

At an earlier point in time (about one week following the onset ofshingles symptoms), it would be expected that T memory cells, althoughnot present in the subject's blood in large concentrations due to thelocalized nature of the VZV infections that cause shingles (i.e.,relatively low blood titers of VZV), would have already recognized theVZV infection and be readily stimulated by the presence of VZV antigen.Accordingly, a T-Cell Memory™ assay was conducted to determine theeffects of the nanofraction, TF XF, Composition A, and Composition B onT memory cells from blood from the same patient as that tested inEXAMPLE 5. The protocol set forth in EXAMPLE 3 was followed, with thefollowing exceptions: VZV vaccine, which had been diluted 1:10, was usedin place of CMV vaccine (for a final, per well dilution of 1:50); andthe sample fractions of EXAMPLE 3 were replaced with the control andimmune modulators used in EXAMPLE 5, with each immune modulator havingbeen diluted to a final, per-well concentration of 100 μg/ml.

The following table sets forth the results of the assay:

TABLE 7 Nano- TF Composi- Composi- Control fraction XF tion A tion BNon-Stimulated (NS) 1 2 13 27 51 Stimulated with VZV 1 5 30 32 69Stimulated with ConA 288

This data is also depicted graphically in FIG. 3.

As expected, transfer factor, which is present in TF XF, stimulatedactivity by the T memory cells. The addition of a small amount of extrananofraction molecules to the transfer factor significantly increasedthe activity of T memory cells, both with and without additional VZVstimulation. Thus, the results of EXAMPLES 5 and 6 confirm that theaddition of extra nanofraction molecules to preparations that alsoinclude transfer factor, even in very small amounts, will down-regulateundesired activity by T-cells (e.g., autoimmunity and associateddisorders, etc.), while improving, or up-regulating, desired T-cellactivity.

Example 7

In another study, which was conducted to determine the abilities ofvarious compositions, including the compositions including transferfactor and extra nanofraction molecules set forth in TABLES 4 and 5, tostimulate natural killer (NK) cell activity against the humanerythroblast leukemia cell line K-562, which is sensitive to NK cells,were evaluated. Accordingly, the NK cells are also referred to herein as“effector cells,” while the K-562 cells are also referred to herein as“tumor cells” and as “target cells.” Specifically, MTT Assay technologywas used, in which 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), which is yellow, is reduced to formazan, which is purple,by reductase enzymes in the active mitochondria of living cells. Justprior to analysis of cytotoxicity, a solution (e.g., dimethyl sulfoxide,sodium dodecyl sulfate (SDS) in dilute hydrochloric acid (HCl), etc.)that will dissolve formazan is added to each well. Spectrophotometry, inwhich the amount of light of a certain wavelength (e.g., a wavelength inthe range of about 500 nm to about 600 nm) absorbed by the solution ineach well is measured, is then used to determine the numbers of livingcells in the assayed wells, relative to the number of living cells inone or more control wells into which no test compositions were added.

The compositions that were evaluated included Transfer Factor Advanced™,available from 4Life Research; Transfer Factor Plus Advanced™, alsoavailable from 4Life Research; Composition A, which includes bothtransfer factor and an elevated amount of nanofraction molecules;Composition B, which includes transfer factor, an elevated amount ofnanofraction molecules, and other ingredients that are believed toenhance immune system activity; nanofraction molecules from bovinecolostrum; nanofraction molecules from chicken eggs; and interleukin-2(IL-2), available under the trade name Proleukin from Chiron of theNetherlands, which is known to enlist NK cells against cancer cells.

Blood was obtained from five healthy donors. Known processes were usedto isolate white blood cells from other constituents of the blood. Knowndensity gradient centrifugation techniques (e.g., using a Histopaque®density gradient available from Sigma-Aldrich Corporation of St. Louis,Mo.) were used to isolate mononuclear cells, including NK cells, fromother types of white blood cells. The mononuclear cells were introducedinto RPMI 1640 growth medium with 10% fetal calf serum (FCS). Equalvolumes of this mixture, including a concentration of about 60,000 whiteblood cells in 100 μl of culture medium, were introduced into differentwells of a standard 96-well plate. Reconstituted samples of the transferfactor and/or nanofraction molecule-containing compositions identifiedabove, each having a concentration of 0.100 mg powder to 1 ml sterile,deionized water, were each added to three different wells containing thewhite blood cells and growth medium, for a total of eighteen wells. Inaddition, 1,000 IU/ml IL-2 were introduced into three positive controlwells containing the mononuclear cell-growth medium mixture. Threenegative control wells included only the mononuclear cell-growth mediummixture, with no immune modulators. Three effector cell-only negativecontrol wells also included only the mononuclear cell-growth mediummixture, while three target cell-only negative control wells includedonly 100 μl of growth medium.

The mononuclear cells were incubated with their respective immunemodulation compositions (except for the three negative control wells) inthe presence of 5% CO₂ at a temperature of 37° C. and 100% humidity for48 hours.

Following incubation, about 30,000 K-562 cells were introduced into eachwell, except for the three effector cell-only negative control wells,that contained mononuclear cells and growth medium. The 96-well plateand the mixtures in its wells were again incubated in the presence of 5%CO₂ at a temperature of 37° C. and 100% humidity for 48 hours.

The MTT solution, having a concentration of 5 mg MTT/ml Henk's salinesolution, was prepared in accordance with known, standard techniques.Twenty microliters (20 μl) of the MTT solution was introduced into eachmononuclear cell-growth medium-tumor cell-containing well of the 96-wellplate. The plate and the contents of its wells were again incubated inthe presence of 5% CO₂ at a temperature of 37° C. and 100% humidity,this time for about four hours.

Following this final incubation, the 96-well plate was centrifuged atabout 1,500 rpm for about five minutes. Thereafter, the supernatant(liquid) was removed from each well, and 150 μl of dimethylsulfoxide(DMSO) was introduced into to each mononuclear cell and tumorcell-containing well. A spectrophotometer was then used to measure theoptical density, at a wavelength of 540 nm, of each cell-containingwell. The measured optical density was then used to determine thecytotoxic index (%) (CI (%)) of the NK cells, as activated by eachtested substance, using the following formula:CI (%)=[1−(OD_(e+t)−OD_(e))/OD_(t)]×100,where OD_(e+t) is optical density of each test well corresponding to atested composition, including the IL-2 of the positive control, OD_(e)is the average optical density of the three effector cell-only negativecontrol wells, and OD_(t) is the average optical density of the threetarget cell-only negative control wells. The CI(%) represents thepercentage of target cells that have been killed by NK cells in eachwell that also contained a tested immune modulation composition. Theresults are presented in the following table:

TABLE 8 Immune Modulation Composition CI (%) Relative Activity TransferFactor Advanced ® 43.1 55 Transfer Factor Plus Advanced ™ 38.5 49Composition A 60.3 77 Composition B 57.9 74 Nanofraction molecules,colostrum 77.9 100 Nanofraction molecules, egg 68.7 88 IL-2 77.0 84

These data, which are also depicted in the chart of FIG. 4, show thatcompositions including nanofraction molecules, particularly those frombovine colostrum, are about as effective as or more effective than IL-2at eliciting NK cell activity against K-562 tumor cells, whilecompositions that include transfer factor from colostrum and eggs andnanofraction molecules from colostrum (i.e., Composition A andComposition B) activate NK cells more effectively than compositions thatlack nanofraction molecules.

By adding as little as 2% more nanofraction molecules, by weight, to acomposition that includes transfer factor, the nanofraction moleculesmay boost action by nonspecific components (e.g., NK cells) of thecell-mediated portion of a subject's immune system, complementing theability of transfer factor to elicit activity by antigen-specificcomponents of the cell-mediated portion of the subject's immune system.

When considered together, the results of EXAMPLES 5 through 7demonstrate that transfer factors regulate and prime T helper cells,which enable a subject's immune system to respond more quickly andefficiently to pathogens and other undesired entities. In addition,EXAMPLES 5 through 7 illustrate that transfer factor may enhance theactivity of T memory cells.

EXAMPLES 5 through 7 also show that the addition of extra nanofractionimmune modulator molecules to compositions that include transfer factormay fortify and enhance the immune modulation (e.g., of T helper cells,T memory cells, and NK cells) of transfer factor and of existingcompositions that include transfer factor.

A method of modulating the cell-mediated immunity of a subject includesadministering (e.g., enterally, parenterally, etc.) a compositionincluding nanofraction molecules to the subject. The nanofractionmolecules may be administered alone, or as part of a composition thatconsists essentially of nanofraction molecules, or they may beadministered with a composition (e.g., a composition such as that setforth in TABLE 4 or TABLE 5) that includes transfer factor.Administration may occur on a regular basis in an effort to maintain anoverall balance in the subject's cell-mediated immunity, or it may beeffected in response to an infection, an autoimmune disorder, tissuetransplant, or another event that affects (activates or suppresses) thecell-mediated immunity of the subject.

Administration of compositions that include nanofraction immunemodulator molecules in accordance with teachings of the presentinvention is believed to modulate cell-mediated immune activity based onphysiological need. For example, undesired cell-mediated immune activity(e.g., autoimmunity, etc.) may be reduced. As another example, theability of T-cells to remove undesirable pathogens, as well as otherundesirable entities, such as cancer cells and other aberrant or mutatedcells, from the body of a subject (e.g., by activating T helper (CD4+)cells, which in turn activate natural killer (NK) cells, by increasingantigen-specific immunity by enabling T memory cells, etc.) may also befocused and enhanced, particularly when transfer factor is administeredwith an additional amount of nanofraction immune modulator molecules.

Although the foregoing description contains many specifics, these shouldnot be construed as limiting the scope of the present invention, butmerely as providing illustrations of some of the presently preferredembodiments. Similarly, other embodiments of the invention may bedevised which do not depart from the spirit or scope of the presentinvention. Features from different embodiments may be employed incombination. The scope of the invention is, therefore, indicated andlimited only by the appended claims and their legal equivalents, ratherthan by the foregoing description. All additions, deletions andmodifications to the invention as disclosed herein which fall within themeaning and scope of the claims are to be embraced thereby.

What is claimed:
 1. A composition comprising an immune modulatingcomponent consisting of: a first fraction of a first quantity of bovinecolostrum, the first fraction having a first upper molecular weightcutoff of about 10,000 Da; a second fraction of a second quantity ofbovine colostrum, the second quantity being separate from the firstquantity, the second fraction having a second upper molecular weightcutoff of about 4,000 Da obtained by separating molecules havingmolecular weights at and below the second upper molecular weight cutofffrom molecules having molecular weights above the second upper molecularweight cutoff; and egg yolk, the first fraction, the second fraction,and the egg yolk being combined with one another.
 2. The composition ofclaim 1, wherein: the first fraction includes transfer factor and immunemodulators; and the second fraction includes immune modulators.
 3. Thecomposition of claim 1, wherein the egg yolk is chicken egg yolk.
 4. Thecomposition of claim 1, wherein the egg yolk is in a dried form.
 5. Thecomposition of claim 4, wherein the first fraction and the secondfraction are in dried forms.
 6. The composition of claim 5, wherein: thefirst fraction makes up about 68% of the total weight of the immunemodulating component; the second fraction makes up about 2% of the totalweight of the immune modulating component; and the egg yolk makes upabout 30% of the total weight of the immune modulating component.
 7. Thecomposition of claim 1, wherein the first fraction is obtained byseparating molecules having molecular weights at and below the firstupper molecular weight cutoff from molecules having molecular weightsabove the first upper molecular weight cutoff.